Navegando por Palavras-chave "Phage Display"
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- ItemSomente MetadadadosConstrução De Biblioteca De Inibidores Proteicos Em Sistema Phage Display Para Seleção De Inibidores Específicos Para Proteases De Larvas De Mosquito Aedes Aegypti(Universidade Federal de São Paulo (UNIFESP), 2017-10-26) Manzato, Veronica De Moraes [UNIFESP]; Tanaka, Aparecida Sadae [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)The mosquito Aedes aegypti is well adapted to urban environment and the major vector of several diseases as dengue, yellow fever, zika and chikungunya flaviviruses. It has been considered a global public health problem because, in the last years, the outbreaks of every 3-5 years of dengue have spread with increased virulence. There is no treatment for these diseases and vaccine has low efficacy, as a result, the prophylaxis remains on vector control but the presence of resistant strains of inseticides and larvicidal in use makes the control a difficult and still unsolved problem. Knowing that the larval stage feeds constantly, we intend to interfere with the larvae development by the inhibition of the digestion proteins. A neutrophil elastase and chymotrypsin inhibitor found in Triatoma infestans eggs named TiPI (Triatoma infestans Pacifastin Inhibitor) had the reactive site (P2-P2’) randomly mutated by the phage display technique which allowed their selection against digestive enzymes of the 4th instar larvae midgut. The selected mutants DNA analyzes evidenced group of 11% possible trypsin inhibitors with Arg or Lys at P1 position, 18.5% possible neutrophil elastase inhibitors with Ala, Leu or Val at P1 and curiously, the major group represented by 47% of the mutants with Gln at P1 and 88.8% with Gln at any mutated position. Fusion phages of ten selected mutants were produced, among them 4 possible trypsin inhibitor, mutants 73 (QRLQ), 154 (LKQL), 161 (KRKQ) and 189 (QRHL), 2 possible chymotrypsin inhibitor 29 (ALAR) and 143 (LLQC), and the 4 most frequently mutants, 4 (SQWH), 5 (QSAM), 6 (HQSL) and 22 (AQVF) and their interactions with proteins of the 4th instar larvae midgut and commercial enzymes were evaluated by Surface Plasmon Resonance. The inconclusive results conduct us to protein expression by Pichia pastoris yeast. The yeast supernatant culture containing recombinant mutants was used in the serine 18 protease inhibition assays. Among the trypsin inhibitors, mutant 73.21 showed high inhibitory activity to trypsin, and the mutant 29.14 inhibits neutrophil elastase. Mutants 4.27, 5.26 and 6.27 presented inhibitory activity for subtilisin A. The results confirm that the TiPI1 phage display library allowed the selection of specific mutants against digestive enzymes of larvae of Ae. aegypti. Our results suggested that these molecules can be use in the development of specific synthetic inhibitors with larvicidal activity for Ae. aegypti.
- ItemAcesso aberto (Open Access)Identificação de inibidores específicos para a protease NS2B-NS3 do flavivírus Zika(Universidade Federal de São Paulo (UNIFESP), 2020-01-30) Santo, Camila Cardoso Di [UNIFESP]; Tanaka, Aparecida Sadae [UNIFESP]; http://lattes.cnpq.br/1168789309568199; http://lattes.cnpq.br/8450399142263600; Universidade Federal de São PauloBesides Dengue, Chikungunya and Yellow Fever Aedes aegypti mosquitoes also transmits Zika virus, and despite vigorous reasearch no vaccine or antiviral is available, and considering no therapy is available a specific Zika virus inhhibitor would allow a faster drug treatment. Zika does not present as severe symptoms as Dengue or Chikungunya, but cases of microcephaly and Guillan-Barré syndrome have been reported, specially microcephaly in fetuses and newborn child, and both consequences of utmost importance. Among viral proteases, NS2B-NS3 represents one of the most studied drug targets due to its role in viral replication, cleaving flaviviral poliproteins into structural and non-structural proteins. In order to find such a specific inhibitor the present work focused on two approaches: 1) identify an inhibitor through kinetic assays of previously characterized molecules from Laboratório de Bioquímica e Biologia Molecular de Artrópodes e Hematófagos (UNIFESP-EPM); and 2) through phage display technique expose the purified recombinant NS2B-NS3 protein to a mutant library previously elaborated. This library, TiPI 1, carry mutations from P2 to P2’, including its reactive site. Regarding the screening, two inhibitors were found: 1) BmKK with Ki of 36.01 nM and 2) Boophilin D1 with Ki of 15.53 nM, in which docking studies suggests interaction between residues D83 from NS2B and reactive site residue K31 from Boophilin D1, the D83 residue was previously described as important for correct folding and therefore activity of NS3 protease. Regarding phage display technique, no unique and redundant mutant sequence was obtained and therefore explored as possible inhibitor, glutamine residues were found throughout the sequences, in P2, P1, P1’ and P2’ positions. Besides no positive results regarding TiPI 1 a new mutant library with the best inhibitor founded, Boophilin D1, should be studied in order to enhance the pursuit of a specific Zika virus inhibitor, enabling even the applications of this method to other flaviviral proteases.
- ItemAcesso aberto (Open Access)Peptídeos Potencialmente Úteis no Tratamento do Câncer de Mama e o Envolvimento de Componentes da Matriz Extracelular na Resistência ao Trastuzumab.(Universidade Federal de São Paulo (UNIFESP), 2011-02-22) Suarez, Eloah Rabello [UNIFESP]; Pinhal, Maria Aparecida da Silva [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)HER2 is a member of epidermal growth factor family of receptors that is an essential mediator of cell proliferation and differentiation. In breast cancer patients, HER2 overexpression is associated with disease aggressiveness, chemo and hormone therapy resistance and poor prognosis. Currently, a HER2 specific monoclonal antibody named trastuzumab, was developed as a treatment for breast cancer; however, there are some reports of resistance to this treatment and it can also cause a high rate of cardiac failure, despite the high cost. The aim of the present study was select specific peptides target to recombinant HER2 protein using a phage display technology. The selected cyclic peptides, called Hercid and Tavelorb, were chemically synthesized. The bacteriophages expressing the specific peptides selected, as well as the synthetic peptides were assayed using different breast cancer cell lines compared to the trastuzumab. Cellular viability, migration and apoptosis/necrosis were evaluated. The results showed that the peptides were able to reduce the cell viability around 50% alone and 85% in association. Tavelorb was able to induce apoptosis/ necrosis in 70% of SKBR3 cells and when associated with Hercid, the effect has increased to 90%. This association decreases tumor cells migration around 85%. The HER2 binding assays showed that the peptides compete with trastuzumab. These peptides co-localize with acidic vesicles around 40-50%, suggesting a possible endocytosis induction and HER2 degradation. The peptides and trastuzumab co-localized with heparan sulfate around 70% suggesting that the binding of these molecules and heparan sulfate could play an important role in antitumoral activity over breast cancer cells. The data propose a potential use of these peptides as an alternative for breast cancer treatment. In addition, we analyzed whether some extracellular matrix components influence trastuzumab treatment. Heparanase-1 (HPSE-1) overexpression effect was analyzed using MCF7 cells stable transfected with HPSE-1 cDNA (MCF7-HPSE-1). The glycosaminoglycans profile, HPSE-1, HPSE-2, Syndecan-1 (Syn-1) and HER2 mRNA expression, HPSE- 1 activity and cell viability were evaluated in different breast cancer cells treated or not with trastuzumab. MCF7-HPSE-1 becomes completely resistant to trastuzumab. HPSE-1 transfection changes the galactosaminoglycans profile of MCF7. Trastuzumab co-localizes in high levels with heparan sulfate (HS) and their binding is necessary to antibody activity. In MCF7 cells, trastuzumab decreases HPSE-1, HPSE-2, HER2 and Syn-1 mRNA expression, while in MCF7-HPSE-1 the antibody increases the mRNA expression of these molecules. SKBR3 cells have the highest expression levels of these molecules, but low HPSE-1 activity, which seems to be determinant to trastuzumab response and modulated by HPSE-2. Our results have demonstrated that an ideal concentration of HS in cell surface and medium, regulated by trastuzumab, is necessary to its action. Secreted HS can sequester trastuzumab, decreasing the antibody amount disposable to interact with HER2 in cell surface. HS secreted could also block HPSE-2, which will not be able to inhibit HPSE-1 activity, contributing for tumor resistance to trastuzumab and supporting tumoral progression. In addition, as the relation HPSE-1/HER2 expression decreases, breast cancer cells sensibility to trastuzumab also decreases. These new insights could be useful when devising strategies for overcoming trastuzumab resistance in HER2 positive cancers.